As reported by Sutherland et al in "Cyclic AMP", Am. Rev. Biochem. 37, 149 (1968), cyclic adenosine monophosphate (C-AMP) has been established as an intracellular "second messenger", mediating many of the actions of a variety of different hormones. According to this theory, first messenger hormones, epinephrine and norepinephrine, influence adenyl cyclase contained at or within cell walls to form intracellularly cyclic AMP from adenosine triphosphate upon receipt of the extra-cellular hormone signal. The formed cyclic AMP in turn functions as a second messenger and stimulates intracellular functions particular to the target cells of the hormone. Cyclic AMP has thus been shown to "activate" certain protein kinases, which in turn produce physiological effects such as muscle contraction, glycogenolysis, steroidogenisis and lipolysis.
More recently, the biochemical functions of guanosine cyclic-3',5' phosphate has been studied. c-GMP has been suggested to be involved, like cAMP, in the cellular mediation of hormone action. A review of the literature with respect to the biochemical function of the cGMP can be found in Miller et al (1972) Biochemistry 12, 5310 herein incorporated by reference and hereinafter summarized. It has been observed that acetylcholine caused an elevation of myocardial cGMP levels when added to the isolated perfused heart. It was further reported that the injection of oxotremorine, a cholimimetic agent, resulted in an increase in acetylcholine in rat brain which was accompanied by a concomitant rise in cGMP levels. Atropine, an anti-cholinergenic agent, prevented this increase in cGMP levels. It was demonstrated that .lambda.M 8-bromo-cGMP was able to mimic the action of the cholinergic agents, carbachol and acetylcholine. This cGMP derivative caused enhanced IgE-mediated release of histamine and of the slow reacting substance of anaphylaxis in human lung fragments. Other studies have shown the administration of acetylcholine resulted in the accumulation of cGMP in heart, brain, and ductus deferens. Finally, the effects of intraperitoneal administration of a variety of 2'-,6- and 8- substituted cAMP, cIMP (inosine cyclic 3',5' phosphate) and cGMP derivatives on blood sugar, blood corticoid levels, heart rate and blood pressure in rats has been reported.
A number of studies have suggested that the physiological action of cGMP are inverse to those of cAMP. The cAMP-stimulated synthesis of .beta.-galactosidase in bacterial cell-free extracts was antagonized by cGMP. It has been shown that dbcGMP and cGMP decreased the rate of the beating of cultured heart cells, in contrast to the acceleration produced by cAMP. Cyclic GMP caused contraction of a rate stomach fundus strip, while increased concentrations of cAMP were associated with smooth muscle relaxation. Renal cortical cGMP levels were decreased in metabolic acidosis, and were increased in alkalosis. Although no changes in cAMP levels were seen during either acidosis or alkalosis, it has been reported that cGMP is suppressed, and cAMP stimulated, the renal cortical production of ammonia from glutamine in vitro. It was also demonstrated that cGMP was capable of labilizing the membranes of lysosomes prepared from sensitized human polymorphonuclear leukocytes, while cAMP stabilized the membrances. Studies indicate that cGMP levels were markedly elevated in the psoriatic lesion, while cAMP levels were decreased. It has also been demonstrated that cholinergic agents of cGMP enhanced the cytotoxicity of antigen-sensitive lymphocytes, while cAMP inhibited cytotoxicity. The phytohemagglutinin-or concanavalin A-induced clonal proliferation of lymphocytes produced a 10-50 fold increase in cGMP levels, while cAMP levels in the cells were essentially unaffected.
It is now well established, as supported in the literature cited in the previously referenced Miller et al article, that increased levels of cAMP are associated with reduced growth rate and induction of differentiation. The growth inhibitory effects of cAMP may be mediated in part by the inhibition of precursor transport into cells. Cyclic GMP antagonizes this inhibition. The available data suggest that cAMP limits growth, possibly by promoting differentiation, while cGMP stimulates growth at the expense of differentiation.
More recently, evidence has been presented that is consistent with the involvement of cGMP in the regulation of prostagladin synthesis and release, Stoner et al, 1973, Proc. Nat. Acad. Sci. USA 70, 3830.
Naturally occurring cyclic nucleotides, cAMP, cGMP and cIMP are degraded, however, in vivo by phosphodiesterase enzymes, which catalyze hydrolysis of the cyclic purine nucleotide to a 5'-monophosphate with a consequent loss of function. It has accordingly been suggested that substituted cyclic AMP, GMP and IMP analogs which are more resistant to phosphodiesterase degradation than the naturally occurring cyclic nucleotide might be administered in aid of lagging cellular processes. Another suggestion has been to enhance the beneifical effects of naturally produced cyclic nucleotides by administering compounds which are capable of inhibiting the undesirable effects of phosphodiesterase enzymes.
Sutherland et al, in Circulation 37, 279 (1968) suggest that the pharmacological effects of theophylline are the result of its ability to inhibit the action of phosphodiesterase enzymes. Theophylline has thus been employed in lieu of the adenyl cyclase-stimulating hormones, epinephrine and norepinephrine, as a heart stimulant following cardiac arrest and in refractory asthma cases as a bronchial dilator. Theophylline, however, does not selectively inhibit phosphodiesterase, but rather gives general stimulation to the central nervous system. Accordingly, the use of theophylline can make the recipient nervous and irritable and can also create cardiovascular effects, i.e., rapid beating. By the same token, theophylline is not as potent a phosphodiesterase inhibitor as is desired and consequently has to be used in larger quantities , which, of course, can further the undesirable effects enumerated above.
Further, due to the apparent opposite cellular effects of cGMP and cAMP, it is desirable to have a class of compounds not only capable of inhibiting phosphodiesterase enzyme, but having the ability to selectively stimulate cGMP protein kinase while not activating cAMP protein kinase.
Recently reported has been the synthesis of a number of 8-alkylthio, 8-arylthio- and 8-alkylamino-cGMP derivatives, along with 8-hydroxy-and 8-bromo-cGMP, some of which have shown the ability to selectively stimulate a purified cGMP-dependent protein kinase from lobster tail but did not activate the cAMP dependent kinase from bovine brain (Paoletti, et al, 1973, Pharmacol. Res. Commun. 5, 87; Miller et al, Biochemistry 12, 5310). Many of these compounds were, in addition, themselves resistant to hydrolysis by cyclic nucleotide phosphodiesterase.
Historically, 8-alkyl purines have been synthesized by Traube cyclization of a 4,5-diaminopyrimidine with the appropriate carboxylic acid fragment (Robins, 1967 in "Heterocyclic Compounds, Vol 8", R. C. Elderfield, Ed., Wiley, New York, N.Y., p. 162). The only 8-acylpurine derivatives known prior to the present invention are 8-acetylcaffeine, 8-propionylcaffeine and 8-acetyltheophylline (Ehrhard and Hennig, 1956, Arch. Pharm. 289, 453), prepared from 8-cyanocaffeine and 8-(1-hydroxyethyl) theophylline, respectively. For the purpose of synthesizing 8-substituted compounds capable of selectively stimulating protein kinase, while resisting and inhibiting phosphodiesterase enzyme reactions, it is desirable to develop methods of introduction of alkyl and acyl groups directly onto existing guanosine 3',5'-cyclic phosphate molecules.
Homolytic alkylation and acylation of heterocyclic system has only recently appeared. Kawazoe, et al, 1972, Chem. Pharm. Bull. 20, 1341, have examined homolytic free radical methylation of guanine, guanosine, and 5'-guanylic acid and found that 8-methylguanine was produced from the action of guanine and t-butyl hydroperoxide, FeSO.sub.4, in the presence of dilute sulfuric acid.
However, there has been no reported synthesis of 8-substituted alkyl or acyl 3',5'-cyclic nucleotides via this method. As previously mentioned, 8-alkyl purines have in the past been synthesized via a Traube cyclization while the few 8-acyl purines synthesized thus far has been accomplished via substitution of other 8-substituted compounds as opposed to direct substitution onto the heterocyclic ring. Where direct substitution to obtain 8-substituted purines has been accomplished, it has been by a nucleophilic or electrophilic displacement reaction.
For example, Robins et al, Ser. No. 474,923, filed May 31, 1974, now abandoned "6,8-Disubstituted-9.beta.-D-Ribofuranosylpurine 3',5'-Cyclic Phosphates", assigned to the same assignee as the present invention, teaches direct nucleophilic or electrophilic substitution of various 3',5' cyclic phosphate nucleotide purine derivatives at the 8 position with such groups as halogens, hydroxyls or amines which readily undergo nucleophilic or electrophilic substitution.